Typhoid fever, caused by the gram negative bacterium Salmonella enterica serovar typhi, is closely associated with poor food hygiene and inadequate sanitation. After ingestion of contamination food and water, Salmonellae are able to penetrate the gut epithelium through specialized microfold cells (M cells) lining the Peyer's patches (Jones et al, 1994, J. Exp. Med., 180, 15-23). After crossing this intestinal barrier, bacteria are able to spread rapidly to visceral tissues, including liver and spleen, where they reside primarily in tissue macrophages. After 5-21 days of incubation, the patients experience fatigue, headache, abdominal pain and fever, constipation or diarrhea. Severe forms may entail cerebral dysfunction, delirium and shock and occasionally intestinal perforation and hemorrhage. S. typhi is a host restricted pathogen causing typhoid fever in humans. However, it is avirulent in susceptible strains of mice. In contrast, other Salmonella serovars, specifically S. typhimurium, S. dublin, S. enteritidis, which are lethal in mice, usually do not cause a disseminated, systemic disease in humans but clinically manifest as gastroenteritis and diarrhea in humans (Fang 1991, Medicine 70, 198-207; Jones 1996, Annu. Rev. Immunol. 14, 533-561). Infection of susceptible mice with S. typhimurium provides a murine model for typhoid fever which bears many similarities to human serovar typhi infection.
According to WHO, the annual global incidence of typhoid fever is 0.3%, corresponding to about 16 million cases, of which approximately 600,000 result in death. Incidence of typhoid fever peaks between the ages of 5 and 12 years in endemic areas. In recent years S. typhi has gradually acquired resistance to oral antibiotics; chloramphenicol, trimethoprim-sulfamethoxazole, ampicillin and tetracycline. Non availability of relevant drugs and rapid development of microbial drug resistance have led to the need of efficacious and affordable vaccines to control typhoid fever. The old inactivated whole cell typhoid vaccines are highly reactogenic, causing high typhoid fever, pain in abdomen, vomiting and diarrhea. Also, these vaccines are only up to 70% effective and the immunity does not persist for more than 3-5 years (Hornick et al 1967, Med. Clin. North. Am. 51, 617-23; Ivanoff et al 1994, Bulletin of the World Health Organization. 72: 957-971).
Immunological protection against typhoid fever requires both cell mediated and humoral responses. Specific cytotoxic T lymphocytes (CTL) have been demonstrated after oral vaccination with live attenuated S. typhi, indicating a role for CTLs in the defense against typhoid fever. Infection is bimodal process determined by host and pathogen. During infection, pathogen as well as host is confronted with various stressful conditions and to protect against these, both increase the synthesis of stress proteins (SPs). Hsps or Sps are highly conserved, ubiquitous and abundant proteins produced by all prokaryotic and eukaryotic cells in response to a variety of physiological insults like heat, hypoxia, nutrient deprivation, oxygen radicals, viral infection, etc. (Morimoto and Milarski, 1990, Stress proteins in Biology and medicine, Cold Spring Harbour, N.Y.; Young 1990, Stress proteins in biology and medicine, Cold Spring Harbour, N.Y.). They ensure survival under stressful conditions that, if left unchecked, would lead to irreversible cell damage and ultimately to cell death. Although they are classified as Sps, Hsps also have essential functions in the cell under normal growth conditions. They are involved in the synthesis, folding, assembly and disassembly of protein complexes and also assist in translocation of proteins from one compartment to another and are often referred to as Molecular chaperones (Ang et al 1991, J. Biol. Chem., 266, 24233-24236; Gething and Sambrook 1992, Nature 355, 33-45, Rothman, 1989, Cell, 59, 591-601). They are divided into a series of families based on their molecular mass in kilodaltons. Hsps 60, 70 and 90 are generally found in cytosol and mitochondria. Gp96 and calreticulum are located in the endoplasmic reticulum.
The Hsps are also involved in several molecular processes of the immune system, such as immunoglobulin chain assembly (Haas 1991, Curr Top Microbiol Immunol 167, 71-92), antigen processing and presentation (Pierce et al, 1991, Curr Top Microbiol Immunol 167, 83-92) and the assembly of functional major histocompatibility complexes I and II (De Nagel and Pierce 1993, Crit. Rev. Immunol. 13, 71-81). Microbial Hsps are dominant antigens for the host immune response to a variety of pathogens including bacteria, fungi, helminthes and protozoan parasites (Young and Elliot 1989, Cell, 59, 5-8) and the immune recognition of Hsp of pathogens serves as a first line of defense. Although the abundance of Hsps may explain why they behave as dominant antigens, it is surprising that the immune system focuses its attention on these proteins which are so highly conserved (bacterial and mammalian Hsps have 50% homology at amino acid level). Most people don't develop dangerous autoimmune responses to self Hsps, although they do posses T cells which recognize these self Hsps, suggesting that these cells are highly regulated (Schawartz 1989, Cell 57, 1073-1081). However, some studies suggest that autoimmune diseases like arthritis may develop through inappropriate cross-reactivity with self Hsp (Young 1990 Ann Rev Immunol 8, 401-420).
The finding that the GroEL complex is involved in bacterial cell wall synthesis, suggests the ready accessibility of these Hsp molecules to antibodies (McLennan and Masters 1998, Nature 392,139). Some studies have suggested the presence of Hsp70 also on the bacterial surface and the heat shock response appears to mediate adhesion to the host cell. Immunologic memory for cross-reactive determinants of conserved Hsp is generated during life, based on frequent re-stimulation by subsequent encounters with microbes with different degrees of virulence. Under these conditions, infection of an individual with a virulent pathogen would enable the already prepared immune system to react quickly before the immune response to more pathogen-specific antigens develops. An immune response to conserved determinants of Hsp shared by different microbes may, furthermore, prevent colonization of the host by microbial pathogens. Several lines of evidence indicate that Hsp also represent unique targets for γδT cells (Fu et al, 1994, J. Immunol 152, 1578-1588)
In mycobacterial infections, reactivity to Hsp predominates, with Hsp60 as an immunodominant target of the antibody and T-cell response in mice and humans. Hsp60-specific antibodies have been detected in patients with tuberculosis and leprosy, and also in mice after infection with M. tuberculosis (Shinnick 1991, Curr Top Microbiol Immunol 167, 145-160). In patients with leprosy or in persons vaccinated with M. bovis BSG, CD4αβT cells specific for the mycobacterial Hsp60 have been found (Mustafa et al 1993, Infect Immun 61, 5294-5301). This finding points to an important role for Hsp60-specific T cells in mycobacterial infection. In vitro stimulation of murine splenocytes and immunization of mice with mycobacterial Hsp60 induced the expansion of CD8αβT cells specific for mycobacterial Hsp60 (Zugel and Kaufmann 1997, Infect. Immun, 65, 3947-3950).
Immune responses to Hsp60 are also frequently found in other microbial infections. In a murine model of yersiniosis, numbers of CD4αβT cells specific for Hsp were increased in infected animals and mediated significant protection against infection with Yersinia enterocolitica when adoptively transferred. Similarly, in infants, levels of antibodies against Hsp60 were significantly increased after vaccination with a trivalent vaccine against tetanus, diphtheria, and pertussis (Del Giudice et al 1993, J Immunol 150, 2025-2032). These findings further suggest that priming of the immune system to Hsp60 is a common phenomenon, occurring at an early stage of life.
Similarly increased antibody levels to Hsp70, have been identified in sera of patients suffering from malaria, leishmaniasis, schistosomiasis, filiariasis, and candidiasis (Shinnick 1991, Curr Top Microbiol Immunol 167, 145-160). In contrast to Hsp60, responses to pathogen-derived Hsp70 seem to be more restricted, sometimes exclusively species specific. Shinnick (1991) demonstrated an important role of the humoral response against Hsp90 in systemic candidiasis. The Hsp90-specific antibodies contributed directly to protection against Candida albicans infection (Matthews and Burnie 1992, Immunol Today 133, 345-348).
Today Hsps are the object of intense work by scientists all over the world as a potential means of vaccines to treat cancer and other diseases. In modern medicine, immunotherapy or vaccination has virtually eradicated diseases such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc. The approach using vaccinations has exploited the ability of the immune system to prevent infectious diseases. Such vaccination with non-live materials such as proteins generally leads to an antibody response or CD4+ helper T cell response (Raychaudhuri and Morrow 1993, Immunol Today 14, 344-348). On the other hand, vaccination or infection with live materials (live cells or infectious viruses) generally leads to a CD8+ cytotoxic T lymphocyte (CTL) response. A CTL response is crucial for protection against cancers, infectious viruses and bacteria. This poses a practical problem, for, the only way to achieve a CTL response is to use live agents which are themselves pathogenic. The problem is generally circumvented by using attenuated viral and bacterial strains or by killing whole cells which can be used for vaccination. These strategies have worked well but the use of attenuated strains always carries the risk that the attenuated reagent may recombine genetically with the host DNA and turn into a virulent strain. Thus, there is need for methods which can lead to CD8+ CTL response by vaccination with non-live materials such as proteins in a specific manner.
Hsps as vaccines are a novel approach to disease prevention. Epitope analysis indicates the presence of multiple B and T cell epitopes in many of these Hsps. They can be used as carriers, vectors and in that regard offer a promising future. There is now substantial evidence that native Hsps (Hsp70, gp96, calreticulum) isolated from tumors can be used as adjuvant free anti tumor vaccines in animal models (Suto and Srivastava 1995, Science, 269, 1585-1588). Srivastava et al (1991, Curr Top Microbiol Immunol 167, 109-123) have demonstrated that immunization of mice with gp96 or Hsp70 isolated from a particular tumor, rendered the mice immune to that particular tumor but not to antigenically distinct tumors whereas corresponding preparations from normal tissues did not grant immunity. Further studies have revealed that Hsps are closely associated with peptides and Hsp depleted of peptides was found to lose its immunogenic activity. The ability of Hsps to potentially bind to the whole cellular peptide repertoire makes them attractive candidates for cancer vaccines. The immunogenic properties of Hsp have been demonstrated in particular for Mycobacterium tuberculosis Hsp70, which has been used successfully as an adjuvant free carrier molecular (Barrios et al, 1992, Eur J Immunol 22, 1365-1372). Suzure and Young (1996, J Immunol 156, 873-879) further demonstrated that in the absence of adjuvants, covalent linking of M. tuberculosis Hsp70 to human immunodeficiency virus type 1 p24 elicited humoral and cellular immune responses to p24, when mice were immunized with this recombinant fusion protein. Most vaccines require adjuvants to provoke effective and protective immune responses, where as Hsp70 fusion proteins induced these immune responses without adjuvants. Most adjuvants used in research cause powerful and unpleasant side effects in humans. Thus only alum, a very weak adjuvant is used in human vaccines.
Hsp fusion proteins elicit antigen specific CTL responses in the absence of adjuvants. Immunization of mice with a soluble fusion protein, consisting of an ovalbumin fragment, a well characterized T cell antigen, covalently linked to mycobacterial Hsp70, induced a strong MHC class I-restricted CD8 T-cell response against a dominant ovalbumin T-cell response against a dominant ovalbumin T-cell epitope and partially protected mice from tumor challenge (Suture et al, 1997, Proc Natl Acad Sci USA 94, 13146-13151). Hsp fusions appear to gain access to MHC class I processing and presentation pathway in a non classical manner. This was unexpected as immunization with soluble proteins, especially in absence of adjuvans, rarely elicit CTL responses. Huang et al (2000, J Exp Med 191, 403-408) showed the ability to elicit CTL is independent of CD4+ lymphocytes, and this function resides in a 200 amino acid domain of Hsp70, concluding that the ability of the fusion proteins to elicit CD8+ T cell does not depend on the Hsps' chaperone properties. They further demonstrated that ovalbumin (OVA) Hsp70 fusion proteins with murine homologue of TB Hsp70 (m Hsp70) also elicited CTL responses equivalent to those generated by mycobacterial Hsp70 fusion protein and was also independent of CD4. This shows that both mycobacterial and murine (self) Hsp70 enhance immune responses.
Numerous studies have documented antibody responses to S. typhi proteins, and the major antigenic components include the somatic 0 antigen (endotoxin, lipopolysaccharide), flagellar H antigen, Vi antigen and outer membrane proteins). In contrast very little is known about host immune response to Salmonella Hsps. The pathogenesis of typhoid fever and the role of various components of the human immune response to Salmonella typhi remain poorly understood. There is little information about S. typhi Hsps and genes which may be involved in virulence or which are important in eliciting a host immune response. The ability of heat shock proteins to chaperone peptides, including antigenic peptides, interact with antigen presenting cells (APC) through a receptor, stimulating antigen (Ag) presenting cells to secret inflammatory cytokines and mediate motivation of dendritic cells, makes them a unique starting point for generation of immune responses. These properties permit the use of Hsps for development of a new generation of prophylactic and therapeutic vaccines against infections agents.
The old inactivated whole cell typhoid vaccines are highly reactogenic, causing high typhoid fever, pain in abdomen, vomiting and diarrhea. In many countries these have been replaced by two currently licensed vaccines: purified Vi polysaccharide parenteral vaccine and Ty 21a, used as a live oral vaccines: purified Vi polysaccharide parenteral vaccine and Ty 21a, used as a live oral vaccine. Ty21a, a mutant S. typhi strain, was isolated by Germanier et at (1982, Bacterial vaccines, pp 419-421. vol 4, New York, USA) and has been used as an orally administered, live, attenuated vaccine. It is in the form of capsules, 3 capsules given orally on alternate days and the capsules need to be swallowed intact. Though the vaccine is effective after 2 years of age, practically a child above 4-6 years of age can swallow capsules. Hence it is recommended after the age of 4-6 years. It is contraindicated in immune compromised host as it is a live vaccine. However, this strain Ty21a has lost an epimerase capable of converting glucose to galactose, a loss resulting in defective synthesis of the polysaccharide component of LPS. As a result Ty21a is not well adapted to survive and multiply in the intestinal tract (Gilman et al, 1977, J. Infect. Dis. 136: 717-23).
These days a parenteral vaccine, which is made of purified Vi capsular polysaccharide, is being widely used. Vi polysaccharide is a well standardized antigen that is effective in a single subcutaneous or intramuscular dose and is safer than whole cell vaccine. But this vaccine can be given only after completing 2 years of age and it confers protection seven days after injection. Besides, it can lead to side effects like pain, swelling, redness, tenderness, etc. and sometimes mild fever lasting for 24 hours. The protective efficacy lasts for 2-3 years in most of the vaccines. Hence one has to revaccinate every three years. Hsps as vaccines are a novel approach to disease prevention.
Srivastava in U.S. Pat. No. 6,410,028 has described the methods of prevention and treatment of cancer and infectious disease by the administration of complexes of human Hsp-antigen molecules to individuals but there is little information about S. typhi proteins and genes which may be involved in virulence or which are important in eliciting a host immune response.
The Hsp based vaccines, unlike other recombinant protein based vaccines, stimulate both humoral and cell mediated immune responses and are currently not available against microbial infections. The present invention, therefore, relates to the development of microbial Hsp based Recombinant vaccine for the prevention of typhoid fever in humans. DnaK (Hsp70) or GroEL protein (Hsp60) of S. typhi either alone or in combination with antigenic molecules is used in the present invention as a vaccine to augment the immune response against S. typhi in mice.